藻胆蛋白荧光探针及其标记

藻胆蛋白是一系列新型的荧光标记探针,具有优良的荧光特性,以藻胆蛋白荧光探针标记抗体还可用于血清可溶性抗原（或抗体）的荧光免疫检测,其标记方法可分为直接法和间接法。结合藻胆蛋白的特点,研究藻胆蛋白的标记方法有助于提高荧光免疫检测的灵敏度。     

Immunoferritin labeling of rabbit spermatozoa

Ferritin-conjugated goat IgG binds nonspecifically to rabbit sperm. This restricts use of ferritin-labeled goat antiglobulins as indirect labels in rabbit sperm antigen localization. Ferritin-conjugated Protein A does not bind nonspecifically to rabbit sperm and is a satisfactory substitute for indirect (“secondary”) labeling of sperm antigens.     

Visualization of the F9 antigen on sperm from normal and T/t-complex mutants by immunoscanning electron microscopy

The antigens defined by conventional syngeneic antiserum against F9 embryonal carcinoma cells were localized on mature sperm using immunolabeling and scanning electron microscopy. Labeling patterns were compared for normal (+ / +) mice and mice bearing recessive t-haplotypes. The results showed that antigens detected by intact anti-F9 antiserum are expressed similarly in all genotypes, except for sperm from mice bearing the t¹²-haplotype where the frequency of labeled cells was reduced. Labeling with the IgM fraction of anti-F9 antiserum was lower on sperm from all t-genotypes examined, with sperm from + /t¹² males showing the most marked reduction. In all cases, the labeling patterns were similar, and included a labeling of the whole sperm head with complete anti-F9 antiserum and a restriction of the label to the postacrosomal region when the IgM fraction was used.     

Short Fixation-Shock Freezing and Freeze-Drying versus Chemical Fixation and Dehydration: Computer Assisted Image Analysis of Morphological Variables and Immunogold Labeling Density on Pituitary Secretory Granules

We have performed a computer assisted image analysis evaluation of the effects of two preparation protocols on morphological variables and immunolabeling density of secretory granules using rat adenohypophysis as a model. Glutaraldehyde (GA) fixation for 2 hr and chemical dehydration was compared with short GA fixation (15 min) followed by cryofixation and freeze-drying (CF-FD). The 2 hr GA fixed specimens showed spherical nuclei and secretory granules regardless of their size, contrasting with the more irregularly shaped nuclei and secretory granules after short GA-CF-FD. In the latter group of specimens a correlation could be found between smaller nuclear areas and more irregular shapes. The differences in morphological variables between the two preparation protocols might be due to a better protein stabilization and a reduced collapse of macromolecules after the 2 hr GA fixation, strengthened by the chemical dehydration. The specific immunolabeling with antiserum to growth hormone was much greater but more varied after short time GA-CF-FD than after long GA fixation. Epon surface topography over the granule area also differed: smooth after short time GA-CF-FD and furrowed after long GA fixation. Our results, demonstrating important differences in morphological parameters and immunolabeling density between two common preparation protocols, seem critical for more reliable interpretation in quantitative immunoelectron microscopy. We also emphasize the need for computer assisted image analysis and measurements in immunoelectron microscopy to ensure objective evaluations.     

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.     

Cell Surface Interactions between Trypanosoma congolense and Macrophages during Phagocytosis In Vitro

ABSTRACT. Trypanosoma congolense bloodstream forms preincubated with a high titer of anti-variant surface antigen (VSG)-specific antibody, a low amount of anti-VSG plus complement-active mouse serum (MS), MS alone, and trypsin were cocultivated with mouse peritoneal macrophages in vitro. Immunofluorescence as well as transmission and scanning electron microscopy revealed that upon attachment to the macrophages' surface, trypanosomes opsonized with anti-VSG/MS formed opsonized filopodia, which were rapidly internalized by the phagocytes. Although these cells attached as frequently as anti-VSG or trypsin-pretreated parasites, the rate of phagocytosis of anti-VSG/MS pretreated trypanosomes was reduced significantly. Trypanosomes pretreated with high antibody titers alone were lysed on the surface of the macrophages before phagocytosis was completed. Parasites opsonized with complement alone adhered only occasionally and were rarely phagocytosed. Trypsin-treated trypanosomes, which served as positive control cells, rapidly attached and remained intact until ingulfment by the macrophages was completed. Untreated control parasites did not attach to the macrophages and were not phagocytosed. Cocultivation of macrophages with anti-VSG/MS-opsonized trypanosomes caused internalization of the flagellum by membrane fusion. Filopodia formation by T. congolense is thus correlated with a marked reduction in phagocytosis even in the presence of only a sublytic antibody titer.     

Specificity of an Antiserum Produced Against Pseudomicrothorax dubius Trichocysts Prepared by a Rapid Isolation Method

ABSTRACT. A rapid method was developed for the isolation of Pseudomicrothorax dubius ciliary and trichocyst fractions which were characterized by SDS-PAGE followed by combined silver and Coomassie blue staining. Antibodies were prepared against the trichocyst fraction and employed to label Lowicryl thin sections of cells. Trichocysts were strongly labeled, as were the surfaces of the plasma and ciliary membranes. Immunoblots of the trichocyst fraction revealed labeling of major bands at 16–29 kD, characteristic of the trichocyst proteins. On immunoblots of the ciliary fraction, approximately eight bands were labeled, including the major cell surface glycoprotein, the immobilization antigen. Ciliary proteins not located on the membrane surface, such as the tubulins, were not labeled. Absorption of the antiserum against fixed P. dubius cells eliminated the cell surface labeling on Lowicryl sections and on immunoblots of the ciliary fraction. The major trichocyst protein bands were as strongly labeled as with the nonabsorbed antiserum. Labeling of several of the minor, higher molecular weight bands of the trichocyst fraction was eliminated, indicating that they are cell surface contaminants. Of the two major structural components of the trichocyst, the shaft and the arms, the antiserum is shown to react nearly exclusively with the shaft proteins on both Lowicryl sections and immunoblots.     

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.     

An Improved Immunostaining and Imaging Methodology to Determine Cell and Protein Distributions within the Bone Environment

Bone is a dynamic tissue that undergoes multiple changes throughout its lifetime. Its maintenance requires a tight regulation between the cells embedded within the bone matrix, and an imbalance among these cells may lead to bone diseases such as osteoporosis. Identifying cell populations and their proteins within bone is necessary for understanding bone biology. Immunolabeling is one approach used to visualize proteins in tissues. Efficient immunolabeling of bone samples often requires decalcification, which may lead to changes in the structural morphology of the bone. Recently, methyl-methacrylate embedding of non-decalcified tissue followed by heat-induced antigen retrieval has been used to process bone sections for immunolabeling. However, this technique is applicable for bone slices below 50-µm thickness while fixed on slides. Additionally, enhancing epitope exposure for immunolabeling is still a challenge. Moreover, imaging bone cells within the bone environment using standard confocal microscopy is difficult. Here we demonstrate for the first time an improved methodology for immunolabeling non-decalcified bone using a testicular hyaluronidase enzyme-based antigen retrieval technique followed by two-photon fluorescence laser microscopy (TPLM) imaging. This procedure allowed us to image key intracellular proteins in bone cells while preserving the structural morphology of the cells and the bone.     

Immunodetection of low concentrations of ovalbumin in cryoprocessed quail oviduct cells by semi-automatic quantitative analysis

Summary— In a previous paper (Quintana et al, Biol Cell 72, (1991) 167–180) we reported the anti-ovalbumin antibody immunolabeling in the cytoplasm of tubular gland cells from cryofixed, cryosubstituted and acrylic-resin embedded quail oviduct. To confirm these preliminary visual observations, we have carried out a semi-automatic quantitative study of immunogold labeling. The quantitative results confirm the previous data. In addition, we found a significantly higher immunolabeling with low temperature Lowicryl K11M embedding procedure as compared with high temperature LR White embedding.